Decreased anti-regenerative effects after spinal cord injury in spry4−/− mice

Highlights

• spry4−/− mice exhibit reduced inflammatory response after spinal cord injury.

• spry4−/− mice exhibit reduced astrogliosis after spinal cord injury.

• Increased neuronal survival after spinal cord injury in spry4−/− mice.

• No difference in lesion size was observed in spry4−/− compared to wildtype mice.

Abstract

Previously, we have demonstrated a role for fibroblast growth factor (Fgf) in spinal cord regeneration in both zebrafish and mouse. We have shown that exogenous Fgf2 treatment attenuates astrocytic gliosis and induces glia cells to become progenitors that undergo neurogenesis as well as differentiating into bipolar astrocytes that support axonal regeneration (Goldshmit et al., 2012, 2014). One of the downstream signaling target genes of Fgf is spry4, which acts as a feedback inhibitor for Fgf signaling. In this study we examined the effects of increased endogenous Fgf signaling, in spry4−/− mice, on the early events that occur after spinal cord injury (SCI). We demonstrate that in spry4−/− mice inflammatory responses, such as tumor necrosis factor α (TNFα) secretion and macrophage/neutrophil invasion into the lesion site are reduced. In addition, astrocytic gliosis is attenuated and neuronal survival is increased. These results further support a pro-regenerative role of Fgf after SCI, and suggest that increased endogenous Fgf signaling after SCI may contribute to functional recovery and therefore presents this pathway as a target for new therapy development.

Introduction

The family of fibroblast growth factors (Fgfs) has been implicated in the regulation of many cellular processes in the central nervous system (Presta et al., 2005). Fgfs have been shown to enhance neurogenesis (Nakatomi et al., 2002, Yoshimura et al., 2003, Ohori et al., 2006) and also to facilitate neuronal survival and neuritogenesis (Abe and Saito, 2001).

Previously we have demonstrated that exogenous Fgf2 treatment after spinal cord injury (SCI) in mice decreased the inflammatory response and astrogliosis near the site of injury. Moreover, Fgf2 treatment increased the number of radial glia and neuronal progenitor cells at the lesion site. In addition, Fgf2 treatment promoted neuronal survival, which improved functional recovery (Goldshmit et al., 2014). Other studies, using brain and SCI models and exogenous Fgf administration, have also shown therapeutic promise, reporting better functional recovery, decreased inflammation and decreased injury volume (Rabchevsky et al., 1999, Rabchevsky et al., 2000, Ruffini et al., 2001, Rottlaender et al., 2011, Kasai et al., 2014). However, some studies also suggest that bFgf may trigger glial reactivity after brain trauma (Eclancher et al., 1996). It was previously shown that Fgf is secreted after SCI along the lesion site and is maintained at high levels for a number of weeks (Tripathi and McTigue, 2008), however, its levels may be partially inhibited by feedback inhibition of its target genes from the Sprouty (Spry) family. Thus the endogenous activity of secreted Fgfs at the wound site and their ability to elicit a pro-regenerative response may well be attenuated by the Spry activity.

Spry proteins act as a negative signaling regulator of receptor tyrosine kinase (RTK) signaling (Dikic and Giordano, 2003, Tsang and Dawid, 2004, Mason et al., 2006) by specific interference with the Ras/MAPK pathway (Gross et al., 2001, Yusoff et al., 2002) A wide range of trophic factors utilize RTK signaling, including Fgf (Hacohen et al., 1998, Impagnatiello et al., 2001), BDNF (brain-derived neurotrophic factor) (Gross et al., 2007), VEGF (vascular epithelial growth factor) (Ayada et al., 2009, Taniguchi et al., 2009a, Taniguchi et al., 2009b), and NGF (nerve growth factor) (Alsina et al., 2012). Besides inhibition through mitogen-activated protein kinase (MAPK) pathway, Spry proteins can suppress PI3-kinase (PI3K)/Akt, PLC (phospholipase C) and other pathways regulated by the Rho family of small GTPases (Taniguchi et al., 2009a, Taniguchi et al., 2009b, Alsina et al., 2012). Spry1, -2 and -4 are expressed in the developing brain (Zhang et al., 2001), and their altered expression has been suggested to disrupt brain development (Suzuki-Hirano et al., 2005). In vitro, downregulation of Spry2 and 4 in hippocampal neurons strongly promotes Fgf2-induced axonal elongation (Hausott et al., 2012). Expression of Spry proteins are directly regulated by Fgf signaling, which in turn modulate the signaling range of Fgf during development and tissue regeneration (Lee et al., 2001, Mason et al., 2006, Faedo et al., 2010, Wang and Beck, 2014).

In a recent study from our laboratory, using a model of SCI in zebrafish, we demonstrated a pivotal role of Fgf, in glial cell proliferation, in morphogenesis and in bridge formation, leading to axonal guidance and regeneration. In addition, we demonstrated that spry4−/− fish regenerated its spinal cord faster than wild-type fish, and facilitated glial bridge formation already after a few days (Goldshmit et al., 2012).

Here we examine whether increased levels of endogenous tyrosine kinase receptor activity, including the Fgf signaling pathway in spry4−/− mice, will mediate similar effects as exogenous delivery of Fgf after SCI. We demonstrate that spry4−/− mice show a significant decrease in the inflammatory response, reduced glial scar formation and increased glial progenitor cells expressing Pax6 around the lesion site. In addition, we observe reduced levels of active caspase 3 that correlate with a significant increase in neuronal survival at the lesion site.